ASO targeting RBM3 temperature‐controlled poison exon splicing prevents neurodegeneration in vivo

Abstract Neurodegenerative diseases are increasingly prevalent in the aging population, yet no disease‐modifying treatments are currently available. Increasing the expression of the cold‐shock protein RBM3 through therapeutic hypothermia is remarkably neuroprotective. However, systemic cooling poses a health risk, strongly limiting its clinical application. Selective upregulation of RBM3 at normothermia thus holds immense therapeutic potential. Here we identify a poison exon within the RBM3 gene that is solely responsible for its cold‐induced expression. Genetic removal or antisense oligonucleotide (ASO)‐mediated manipulation of this exon yields high RBM3 levels independent of cooling. Notably, a single administration of ASO to exclude the poison exon, using FDA‐approved chemistry, results in long‐lasting increased RBM3 expression in mouse brains. In prion‐diseased mice, this treatment leads to remarkable neuroprotection, with prevention of neuronal loss and spongiosis despite high levels of disease‐associated prion protein. Our promising results in mice support the possibility that RBM3‐inducing ASOs might also deliver neuroprotection in humans in conditions ranging from acute brain injury to Alzheimer's disease.

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Referee #2 (Remarks for Author): The paper by Preußner et al. describes the use of an ASO to alter the amount of RBM3 produced the MOE chemistry is used which is the same as that used in the FDA approved drug Spinraza used to treat SMA. The RBM3 is a gene that is associated with a response to cold shock and acts as a neuroprotectant it is also indicated to increase cell proliferation and has been implicated as a protooncogene. The ASO used is conserved in humans and mice which is useful for translation (Namely M2D). The authors do show effectiveness in the Prion model and as such this is useful information. The main factor that should be toned down while this certainly is an interesting target neuroprotection has a relatively poor record in translation so at the moment it is relatively unclear how well this will translate for neurodegenerative disorders. The authors for instance only give the positive expel of spinraza and do not discus the negative ASO trials in Huntington's. My specific comment are as follows 1) Doses of 100ug to 300ug of ASO seem relatively high compared to SMA treatments this does get somewhat confusing as the mice in SMA were tested at 20ug/g when given through the CSF whereas the clinical dose in patients is 12 mg so an infant of 3000g so .012g/ 3000g which is 4ug/ g. It just should be clear how the doses compare you can usually also see a dose response. The amount given to the mouse here is 300ug assuming a weight of 23grams this is 13ug/g. So, in a similar range to what was given to the mouse but lower than the dose used in humans.
2) The authors use w.p.i for post inoculation is a little confusing as it could also be post injection it might help to clarify that all time points are relative to inoculation with Prion.
3) The RBM3 levels could change with Prion infection as it is a stress can a comparison between a Prion infected and noninfected animal be given so as to ensure the level of change is not just due to infection I presume not as the untreated Prion infected animals are a comparison group.
4) The authors indicate an overly positive view on the potential of RMB3 for treatment of a series. diverse conditions, from acute treatment of neonates through to cardiac surgery, stroke and head injury in adults, to longer term neuroprotection in degenerative disorders. First it is not really clear in most of these situations how much protecting a neuron is going to do if it is alive and not functioning. Second neuroprotection has not as yet really proved very effective in the clinic. So yes it is what is wanted but we need to waith to really see this is the case. In particular the authors should discuss the negative results in ASO clinical trials as well as the positive. In the case of Huntingtons the ASOs to knockdown Huntingtons appeared to work in mice but failed in clinical trials. The inatial trials of ASOs in myotonic also failed most likely due to inefficient uptake by muscle. Both these programs used MOEs. Often the exact reason for failure can be difficult to determine but the positive of SMA should be balanced with some of the negatives in discussion. It is also likely that large animal studies of the ASO will need to be done so as to ensure safety and no adverse effects or cancer induction. The licensing of the ASOs in ALS does not mean there are no problems ahead to overcome. The discussion needs to be more balanced.
5) The ASO sequences are given in a supplementary table but the complete sequence of Exon 3b could be given in the figures with a bar above indicating the critical oligos in the case of the ASOs a numbering system from the intron was used this form of naming could also be added to the M2 name ie M2 32-47 (this is not the correct number for M2 ASO just an example of the numbering system) ie the exon base number from the 5 prime boundary this will be useful in reading the text. It is also quite lucky that the sequence of M2 is conserved and would be useful to know if it is conserved in all larger species that could be used for tox ie pig dog and monkey.

Referee #3 (Comments on Novelty/Model System for Author):
This is a smart study, very well described, from an initial phase of ASO design, followed by an in vitro analysis and validation; and with a final in vivo testing. The authors have also a strong background the in the study of RBM3 that supports the results and the final conclusion of the analysis.
Referee #3 (Remarks for Author): The article submitted by Marco Preußner et al to EMBO Molecular Medicine journal, and titled "ASO targeting temperaturecontrolled RBM3 poison exon splicing prevents neurodegeneration in vivo" describes the potential utility of the ASO therapeutic strategy to induce the increase of RBM3 expression, without the natural RBM3-inductor; cooling. The authors report that a single administration of ASO to exclude the poison exon, results in long-lasting increase of RBM3 expression in cells and mouse brains. In addition, in prion-diseased mice, this treatment leads to remarkable neuroprotection, with prevention of neuronal loss. The authors conclude that this novel approach used to stimulate the expression of RBM3 open the possibility to explore the beneficial effect of this cold-dependent protein to induce brain protection in acute brain injury or Alzheimer's disease, by avoiding the sides effect of hypothermia. This is a smart study, very well described, from an initial phase of ASO design, followed by an in vitro analysis and validation; and with a final in vivo testing. The authors have also a strong background the in the study of RBM3 that supports the results and the final conclusion of the analysis. Chemical agents or agonists to induce the pharmacological expression of RBM3 have been also suggested in other articles without good results so far, and this new approach is a smart alternative strategy to explore the therapeutic use of RBM3.
Some minor points related with the future application of ASO-RBM3 could be included in the discussion of the manuscript.
A recent study published in stroke (J Clin Med. 2022 Feb 11;11(4):949) and supported by previous studies (J. Cereb. Blood Flow Metab. 2019;39:2355-2367Neuroscience. 2015;305:268-278.), suggests that the recombinant form of the hormone FGF21 could be used as an external therapy to modulate the expression of RBM3. This strategy seems more translational than ASO therapy.
The use of ASO has been tested in experimental animals as a single intracerebroventricular injection that is no so invasive, but thinking in the potential application in old patients with Alzheimer or stroke (for instance), alternative and less invasive routes of administration are more convenient.
Based on the previous study published by same authors (Life Sci Alliance. 2021 Feb 9;4(4):e202000884); Was TrkB signalling analysed to guarantee that the protective effect was mediated by RBM3?

Response to Referees
We thanks the Reviewers for their constructive and insightful comments. The Referees' comments are reproduced in full. Our responses follow each point in blue text.

Referee #2 (Comments on Novelty/Model System for Author):
Well the Novelty and medical impact would be improved by knowing you could get good distribution in a larger animal and that no tox arose.
We agree with the Referee and larger animal studies will follow this one. We mention this is the discussion (p7). We saw no signs of toxicity for ASO M2D in mice up to 9 weeks after inoculation (throughout the duration of the experiment). We have added a line to this effect on p6.

Referee #2 (Remarks for Author):
The paper by Preußner et al. describes the use of an ASO to alter the amount of RBM3 produced the MOE chemistry is used which is the same as that used in the FDA approved drug Spinraza used to treat SMA. The RBM3 is a gene that is associated with a response to cold shock and acts as a neuroprotectant it is also indicated to increase cell proliferation and has been implicated as a protooncogene. The ASO used is conserved in humans and mice which is useful for translation (Namely M2D). The authors do show effectiveness in the Prion model and as such this is useful information.
The main factor that should be toned down while this certainly is an interesting target neuroprotection has a relatively poor record in translation so at the moment it is relatively unclear how well this will translate for neurodegenerative disorders. The authors for instance only give the positive expel of spinraza and do not discus the negative ASO trials in Huntington's. My specific comment are as follows 1) Doses of 100ug to 300ug of ASO seem relatively high compared to SMA treatments this does get somewhat confusing as the mice in SMA were tested at 20ug/g when given through the CSF whereas the clinical dose in patients is 12 mg so an infant of 3000g so .012g/ 3000g which is 4ug/ g. It just should be clear how the doses compare you can usually also see a dose response. The amount given to the mouse here is 300ug assuming a weight of 23grams this is 13ug/g. So, in a similar range to what was given to the mouse but lower than the dose used in humans.
We agree. In fact, for the prion study we used 200ug, which is around 8ug/g. This remains within the range for application in humans cited by the reviewer. We have clarified this in the text on p6.
2) The authors use w.p.i for post inoculation is a little confusing as it could also be post injection it might help to clarify that all time points are relative to inoculation with Prion.
We have made this clear in the text on p6.
3) The RBM3 levels could change with Prion infection as it is a stress can a comparison between a Prion infected and non-infected animal be given so as to ensure the level of change is not just due to infection I presume not as the untreated Prion infected animals are a comparison group.
16th Jan 2023 1st Authors' Response to Reviewers RBM3 shows non-statistically significant increase in prion infection (see Peretti et al, 2015, Nature 518: 236-239), but the key control here is the effect on RBM3 of the ASO M2D vs control ASO in disease. In addition, control ASOs do not increase RBM3 levels in wild type mice (see Fig. 3G).

4)
The authors indicate an overly positive view on the potential of RMB3 for treatment of a series. diverse conditions, from acute treatment of neonates through to cardiac surgery, stroke and head injury in adults, to longer term neuroprotection in degenerative disorders. First it is not really clear in most of these situations how much protecting a neuron is going to do if it is alive and not functioning. Second neuroprotection has not as yet really proved very effective in the clinic. So yes it is what is wanted but we need to waith to really see this is the case. In particular the authors should discuss the negative results in ASO clinical trials as well as the positive. In the case of Huntingtons the ASOs to knockdown Huntingtons appeared to work in mice but failed in clinical trials. The inatial trials of ASOs in myotonic also failed most likely due to inefficient uptake by muscle. Both these programs used MOEs. Often the exact reason for failure can be difficult to determine but the positive of SMA should be balanced with some of the negatives in discussion. It is also likely that large animal studies of the ASO will need to be done so as to ensure safety and no adverse effects or cancer induction. The licensing of the ASOs in ALS does not mean there are no problems ahead to overcome. The discussion needs to be more balanced.
We thank the Referee for bringing up these points. We have addressed these comments for a more balanced discussion on p7 and specifically mention the Huntingtons trial and the need for additional studies in larger animals.
5) The ASO sequences are given in a supplementary table but the complete sequence of Exon 3b could be given in the figures with a bar above indicating the critical oligos in the case of the ASOs a numbering system from the intron was used this form of naming could also be added to the M2 name ie M2 32-47 (this is not the correct number for M2 ASO just an example of the numbering system) ie the exon base number from the 5 prime boundary this will be useful in reading the text. It is also quite lucky that the sequence of M2 is conserved and would be useful to know if it is conserved in all larger species that could be used for tox ie pig dog and monkey.
Sequences for human and mouse E3a are too large to present them within one clear Figure, so we provide these sequences in Table S1. As we would prefer to stick to our nomenclature, the exact binding positions within exon 3a are now provided in Table S2. Additionally, we provide an alignment of human, chimp, rhesus, dog, pig and mouse M2 sequences (in Figure EV4A) and highlight the ASO binding sites within these alignments. This reveals 100% conservation for the M2D target site across all these species.

Referee #3 (Comments on Novelty/Model System for Author):
This is a smart study, very well described, from an initial phase of ASO design, followed by an in vitro analysis and validation; and with a final in vivo testing. The authors have also a strong background the in the study of RBM3 that supports the results and the final conclusion of the analysis.

Referee #3 (Remarks for Author):
The article submitted by Marco Preußner et al to EMBO Molecular Medicine journal, and titled "ASO targeting temperature-controlled RBM3 poison exon splicing prevents neurodegeneration in vivo" describes the potential utility of the ASO therapeutic strategy to induce the increase of RBM3 expression, without the natural RBM3-inductor; cooling.
The authors report that a single administration of ASO to exclude the poison exon, results in longlasting increase of RBM3 expression in cells and mouse brains. In addition, in prion-diseased mice, this treatment leads to remarkable neuroprotection, with prevention of neuronal loss. The authors conclude that this novel approach used to stimulate the expression of RBM3 open the possibility to explore the beneficial effect of this cold-dependent protein to induce brain protection in acute brain injury or Alzheimer's disease, by avoiding the sides effect of hypothermia. This is a smart study, very well described, from an initial phase of ASO design, followed by an in vitro analysis and validation; and with a final in vivo testing. The authors have also a strong background the in the study of RBM3 that supports the results and the final conclusion of the analysis. Chemical agents or agonists to induce the pharmacological expression of RBM3 have been also suggested in other articles without good results so far, and this new approach is a smart alternative strategy to explore the therapeutic use of RBM3.
Some minor points related with the future application of ASO-RBM3 could be included in the discussion of the manuscript.
A recent study published in stroke (J Clin Med. 2022 Feb 11;11(4):949) and supported by previous studies (J. Cereb. Blood Flow Metab. 2019;39:2355-2367Neuroscience. 2015;305:268-278.), suggests that the recombinant form of the hormone FGF21 could be used as an external therapy to modulate the expression of RBM3. This strategy seems more translational than ASO therapy.
We have included this approach and reference the 2019 study in the discussion on p7.
The use of ASO has been tested in experimental animals as a single intracerebroventricular injection that is no so invasive, but thinking in the potential application in old patients with Alzheimer or stroke (for instance), alternative and less invasive routes of administration are more convenient.
We agree, however, our study is a proof-of-principle of the approach and alternative delivery methods may become available. We do include a sentence highlighting the pursuit of alternative means of induction as future strategy on p7.
Based on the previous study published by same authors (Life Sci Alliance. 2021 Feb 9;4(4):e202000884); Was TrkB signalling analysed to guarantee that the protective effect was mediated by RBM3?
TrkB signalling is upstream of RBM3 splicing, initiated via BDNF binding of the receptor and induces RBM3 through a signalling cascade. This ASO is acting very much downstream of this, at the level of the pre-mRNA of RBM3 in the basal state (independent of activation of TrkB or other inducers) and we thus did not analyse TrkB signalling. We agree that it will be an interesting research direction to connect TrkB signalling with RBM3 AS-NMD in future research. Congratulations on a great revision! Overall, the referees have been positive. However, referee 2 has asked for a minor inclusion to the discussion that we would like you to include in an updated version.
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